Solvent and solvent proton dependent steps in the galactose oxidase reaction

Abstract

Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl beta-galactopyranoside by O2 was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable delta H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D2O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D2O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of kcat vs. mole fraction D2O was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN- inhibition was also examined. The Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN- was also deprotonating above pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic Isotope Effects as Probes of the Mechanism of Galactose Oxidase

“…The reactions with the β-haloethanols leads to rapid enzyme inactivation by either reduction of the radical center by one electron (reversible) or irreversible halogenation or alkylation of the protein. A solvent isotope effect (k H 2 O /k D 2 O ) 1.55) has also been reported for the oxidation of galactose catalyzed by GO (13).…”

Ligand interactions with galactose oxidase: mechanistic insights

“…The energy range of the d -* d spectra of the cupric center rules out an effective tetrahedral copper geometry in galactose ox-idase that has been suggested on the basis of the long Cu-water distance observed in the crystal ( 14). The relatively weak perturbation of the metal d-orbitals in a tetrahedral copper environment is associated with ligand field transitions below 8,000 cm-' (beyond 1,250 nm) (24).…”

“…Note that a coordinated water is a structural analog for bound alcohol in the substrate complex, and our results suggest that deprotonation ofthe coordinated hydroxyl group occurs as a substrate activation step in catalysis. Solvent kinetic isotope effects in the turnover reaction previously have been interpreted in terms of a rate-limiting ionization of coordinated water in the active site (24), but this neglects the rapid exchange of substrate hydroxyl protons in deuterated water. The results are equally consistent with deprotonation of coordinated substrate, and we propose that substrate rather than water ionization is the step sensitive to solvent deuteration.…”

Mechanism and Specificity of Human α-1,3-Fucosyltransferase V

“…The proton inventory technique has been used widely to characterize the transition-state nature of many enzymes (Venkatasubban & Schowen, 1985). Enzymes as varied as HIV protease (Hyland et al, 1991), β-galactosidase (Selwood & Sinnott, 1990), phosphotyrosyl protein phosphatase (Zhang & Etten, 1991), and galactose oxidase (Driscoll & Kosman, 1987) have been studied by this technique. The proton inventory of FucT V is consistent with only one proton "in flight" in the transition state through statistical analysis of the fit of the data to the Gross-Butler equations for one-or two-proton transfer reactions (eqs 5 and 6).…”